American Society for Microbiology Acta Medica Okayama 2165-0497 14 11 2026 A novel SNP-based multiplex PCR profiling divulges contemporary genetic patterns in Haitian variant Vibrio cholerae O1 clinical isolates e02661-25 EN Prosenjit Samanta Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Priyanka Ghosh Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Goutam Chowdhury Collaborative Research Centre of Okayama University for Infectious Diseases, ICMR-National Institute for Research in Bacterial Infections Sreeja Shaw Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Arindam Naha Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Deboleena Roy Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Ayumu Ohno Collaborative Research Centre of Okayama University for Infectious Diseases, ICMR-National Institute for Research in Bacterial Infections Shin-ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Thandavarayan Ramamurthy Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Asish Kumar Mukhopadhyay Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections The massive cholera outbreak in Haiti in 2010 has drawn the attention of both medical professionals and public health officials worldwide. The hypervirulent Vibrio cholerae O1 El Tor strains responsible for this outbreak were not only confined to Haiti but also spread quickly to other cholera-prone regions. Therefore, tracking and understanding these strains in endemic areas have become crucial for timely and effective treatment strategies. The presence of specific genetic mutations in key virulence-related genes\ctxB (cholera enterotoxin subunit B), tcpA (toxin-coregulated pilin), and rtxA (repeats-in-toxin)\makes these Haitian strains distinctive. These genes contain single nucleotide polymorphisms, which act as unique markers for identifying the Haitian variant. However, current detection methods using allele-specific simplex PCRs are time-consuming and can delay critical outbreak responses. To address this, we developed new Haitian allele-specific primers targeting rtxA and tcpA and combined them with an existing ctxB-specific PCR into a single multiplex PCR assay. We tested this new multiplex PCR on 250 clinical V. cholerae O1 isolates collected from diarrheal patients in Kolkata, India, between 2002 and 2019. Interestingly, we noticed that the first appearance of Haitian tcpA, rtxA, and ctxB alleles was in 2003, 2004, and 2006, respectively, years before the Haitian outbreak occurred. Our results matched perfectly with the conventional simplex PCRs and demonstrated 100% sensitivity and specificity. Given the global spread of these Haitian-like V. cholerae strains, this multiplex PCR provides a fast, reliable tool for identifying cholera infection and gaining valuable epidemiological insights. No potential conflict of interest relevant to this article was reported.